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Amplification-free ...
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Höijer, IdaUppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik
(author)
Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity
- Article/chapterEnglish2020
Publisher, publication year, extent ...
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2020-12-01
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Springer Science and Business Media LLC,2020
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electronicrdacarrier
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LIBRIS-ID:oai:DiVA.org:uu-428344
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https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-428344URI
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https://doi.org/10.1186/s13059-020-02206-wDOI
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Language:English
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Summary in:English
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Subject category:ref swepub-contenttype
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Subject category:art swepub-publicationtype
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BACKGROUND: One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro.RESULTS: The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites.CONCLUSIONS: Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.
Subject headings and genre
Added entries (persons, corporate bodies, meetings, titles ...)
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Johansson, JosefinUppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab(Swepub:uu)josjo523
(author)
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Gudmundsson, Sanna,1989-Uppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab,Program in Medical and Population Genetics, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA; Division of Genetics and Genomics, Boston Children’s Hospital, Boston, MA, USA(Swepub:uu)sangu463
(author)
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Chin, Chen-Shan
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Bunikis, IgnasUppsala universitet,Science for Life Laboratory, SciLifeLab,Institutionen för immunologi, genetik och patologi(Swepub:uu)ignbu776
(author)
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Häggqvist, SusanaUppsala universitet,Institutionen för immunologi, genetik och patologi,Science for Life Laboratory, SciLifeLab(Swepub:uu)susha415
(author)
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Emmanouilidou, AnastasiaUppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab(Swepub:uu)anaem996
(author)
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Wilbe, MariaUppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)marwi726
(author)
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den Hoed, Marcel,1980-Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)marde358
(author)
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Bondeson, Marie-Louise,1960-Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)malobond
(author)
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Feuk, LarsUppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)larsfeuk
(author)
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Gyllensten, UlfUppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)ulfgyll
(author)
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Ameur, AdamUppsala universitet,Institutionen för immunologi, genetik och patologi,Science for Life Laboratory, SciLifeLab,Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia(Swepub:uu)adame789
(author)
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Uppsala universitetScience for Life Laboratory, SciLifeLab
(creator_code:org_t)
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In:Genome Biology: Springer Science and Business Media LLC21:11465-69061474-760X
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